High-efficiency oblique illumination module for fluorescence excitation and detection

ABSTRACT

Disclosed is a module for efficiently exciting and detecting fluorescence. In a disclosed preferred embodiment, excitation lamp modules with multiple solid-state light sources shone at different oblique angles, such that multiple-beams converge in a single plane, are combined with appropriate emission filters that block the excitation light wavelengths while passing the emission wavelengths. A multitude of such modules can be combined on a slider with precise registration via a mating structure, and can be accurately positioned in front of an observation point, such as a microscope&#39;s objective lens, external to the microscope, via a novel magnetic detent system, allowing for the rapid switching between the analysis of different fluorophores.

BACKGROUND OF THE INVENTION

There are many analytical methodologies that rely on the excitation anddetection of fluorescence. For example, it is possible to determinewhere and when genes are expressed in a developing organism using thetechnique of fluorescence microscopy. A hybrid gene can be created thatcombines the regulatory elements of a gene of interest with the geneticcoding sequences of a fluorescent protein product (a fluorophore) gene.In this case, the hybrid gene is used to create a transgenic organismsuch that the fluorophore will be expressed in the same cells, at thesame times, as a particular endogenous gene of interest, so that one maylearn where and when that gene is expressed. In order to accomplish thisanalysis, bright light of a particular wavelength must be shone upon thesample (this light is sometimes refined using an “excitation filter”),and the resulting excitation wavelengths must be blocked from theobserver such that different and dimmer fluorescent wavelengths can beobserved (sometimes using an “emission filter”). A description of thistechnology and details about a multitude of fluorophores can be found inCHUDAKOV, D. M. et al., “Fluorescent Proteins and Their Applications inImaging Living Cells and Tissues” in the Physiological Reviews of theAmerican Physiological Society, 2010, which is hereby incorporated byreference in its entirety. A similar method and apparatus can be used todetect the composition of minerals and gemstones, and for a variety ofother applications.

The traditional method for accomplishing fluorescence microscopy uses avery bright, polychromatic light source. The polychromatic light sourceis then filtered so as to block all of the wavelengths other than thespecific ones needed to excite fluorescence, thus most of the light iswasted and discarded. The polychromatic light source is typically an arclamp that consumes a lot of power, generates a lot of heat, may containtoxic materials such as mercury, and is short-lived. The filteredexcitation light is then injected into the optical path, with anexpensive, angled dichroic mirror in a method termed “epi-illumination”.To accomplish this, an excitation filter, and an emission filter areinserted into the faces of a box to form a right angle, with a dichroicmirror between them. This arrangement of two filters and a mirror iscalled an “epi-fluorescence cube”. Some losses are realized as theexcitation light reflects off the mirror, at an angle, and furtherlosses are realized as the emitted fluorescence must pass through thedichroic mirror, at an angle, to be seen by the observer. One convenientfeature of epi-fluorescence is that it is possible to switch betweendifferent colored fluorophores quickly by sliding or rotating one cubeout of the light path and another cube into the light path. Reichmandiscloses such a system in U.S. Pat. No. 6,414,805B1 “Reflected-lighttype fluorescence microscope and filter cassette used therefor”. Thissystem has several shortcomings. It relies on an inefficient, wastefulpolychromatic light source, and it requires an expensive microscopespecially designed for access into the light path to accommodate thecube changer system, and a cube changer system that fits a particularmodel of microscope. Another challenge and shortcoming of moveableoptical elements (for example the cubes) is that they must be positionedprecisely, over and over as the user switches back and forth betweenthem. This typically requires some kind of a detent system. Traditionaldetent systems use spring-loaded elements that scrape along a surfaceuntil they fall into a depression at the appropriate point. Thus, thesesystems are subject to wear. Additionally, if too much force is used,such that a detent point is passed, there is nothing present to preventthe linear slider or rotary slider (turret) from moving much further.

As solid-state light sources, such as Light Emitting Diodes (LEDs) havebecome powerful and practical, prior art has used these longer-lived,less expensive light sources in place of the arc lamps. With LEDs, oneoption is to use a power-hungry multitude of polychromatic “white” LEDsaimed inefficiently into an expensive light pipe, and from there intothe epi-fluorescence cube located within the special microscope. Thishas all of the problems described above except that LEDs are long-lived.Alternatively, light from a single LED with an appropriate narrow colorspectrum can be injected into the optical path via epi-illumination. Forexample, in U.S. Pat. No. 7,502,164B2, Lytle discloses a “Solid statefluorescence light assembly and microscope”. This system combines onesingle-colored LED into each of several changeable epi-fluorescencecubes. Unfortunately, this system requires the use of a highlyspecialized microscope that both allows for epi-illumination, andadditionally has provisions for powering the LEDs built into themicroscope. Furthermore, the available geometry to illuminate thespecimen by epi-illumination limits illumination to one LED per cube.The prior art also contains examples of using one or more LEDs, of asingle color, to illuminate the specimen obliquely. For example Mazelteaches of the “Stereo Microscope Fluorescence Adapter,” on the web pagewww.nightsea.com/products/stereomicroscope-fluorescence-adapter/. Thisis a meager device that shines an LED's light on the specimen from agoose-neck lamp and blocks the excitation light with a manuallyinstalled filter plate. In US20070153372A1, Mazel discloses a“Fluorescence illumination method and apparatus for stereomicroscopes”.This system uses only a single type of ultraviolet light LED to excitefluorescence that cannot be changed quickly and the fact that thisdevice can mix in white light is not relevant for fluorescencedetection. The system has no provisions for quickly analyzing multiplefluorophores that require provisions for different excitation andemission spectra. A significant drawback to prior single-colored LEDdirect illumination approaches is that it is nontrivial to switchbetween analyzing different fluorophores because LEDs, excitationfilters, and emission filters, must all changed simultaneously, andprior art does not provide for this. Another problem is that individualLEDs are often not powerful enough to excite a useful level offluorescent signal.

SUMMARY OF THE INVENTION

The current invention is a system that solves problems associated withusing electromagnetic radiation sources, for example LEDs, to excite andanalyze fluorescence. The system is a method and associated devices topractice the invention. A key feature of the invention is that it takesplace independently and/or externally to any other piece of equipment.For example, if a microscope is used to observe fluorescence, theinvention is located outside of the microscope and not dependent oninterrupting the optical path of the microscope to excite the specimenor to filter the emission from the specimen. Elements of the inventionmay include the ability to rapidly and easily change between thecombined excitation and analysis of different fluorophores via differentmodules and/or the ability to more intensely excite fluorophores throughthe use of modules producing multiple intersecting beams ofelectromagnetic radiation that converge at the specimen thus increasingexcitation of a bona fide signal in the plane and location of thespecimen while not increasing the background noise signal in otherplanes or locations.

Useful features that can contribute to the invention include the use ofmagnets to accurately position the modules via a moving element and theuse of magnets to attach the modules to the moving element so that auser can rapidly configure the system to excite and analyze differentfluorophores in different orders without the use of any tools.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 represents one preferred embodiment of the illuminator. (1) is anenclosure pertaining to Light Emitting Diodes (LEDs) (101), (102), and(103). The light rays from these LEDs are collimated by optics (104),(105), and (106), respectively, and filtered by filters (19), (108), and(20), respectively. The resulting light beams, (110), (111), and (112),respectively, intersect and add at the observation plane (114), but notin planes in front of (for example, (113)) or behind (for example (115))the sample plane of interest.

FIG. 2 represents one preferred embodiment of a High-efficiency ObliqueIllumination System for Fluorescence Excitation and Detection for amulti-fluorophore fluorescence microscope system. Multi-LED lampenclosure (1), with excitation filters (19) and (20) is furtherassembled to emission filter (2); similarly, multi-LED lamp enclosure(3), with excitation filters (21) and (22), different from (19) and(20), is further assembled to a different emission filter (4). Thesecombined assembly modules of excitation light sources with emissionfilters are detachably attached to movable chassis (slider) (5). Astereo-microscope is represented by main body (12A), eyepieces (12B) andobjective lens (12C). Slider-holder (10) positions the slider (5) in theoptical path of the microscope. Through the use of handles (17) and(27), slider (5) can be translated in directions (18) and (28) by eitherpushing action or pulling action, such that either lamp/filterassemblies (1) plus (2) or (3) plus (4) are positioned below the opticalpath of the microscope and electrical contacts are established toenergize the lamps within them via contacts (13) plus (14) or (15) plus(16), respectively. Optimal positioning of the slider is insured by theattractive force of slider magnets (6), (7), and (8) with stationaryreference magnet (9) located in the slider-holder (10). The multi-LEDlamps have beams aimed to converge at point (23) in the focal plane ofthe microscope. The spot (23) is also covered by the matching emissionfilter. Pivot screws (24) and (25) allow lamp enclosures (1) and (3),respectively, to pivot to precisely illuminate spot (23) when in theactive position. For example, lamp enclosure (1) is in the activeposition when slider magnet (6) aligns with stationary reference magnet(9). Not shown in this figure, because they are blocked from view byslider (5), are adjustable jack screws coming toward the slider fromenclosures (1) and (3) The heads of these jack screws hit slider (5) sothat the tilt angles of lamp enclosures (1) and (3) to can be adjustedto precisely illuminate spot (23) when in the active position.

FIG. 3 is a drawing of an alternate embodiment of the invention withmultiple lamps of various colors aimed at the sample of interest,orthogonally with respect to the axis of observation. A microscope,represented by its body (12A) and eyepiece tubes (12B) is focused at alocation (209) where a specimen of interest would be in the center ofthe field of view of a stage plate (200). Excitation sources (203) and(204) project electromagnetic energy along intersecting beams (210) and(211) that intersect at the sample location (209). These beams are bothat wavelengths that excite a particular fluorophore. Similarly,excitation sources (205) and (206) are both at wavelengths that excite asecond fluorophore, and excitation sources (207) and (208) are both atwavelengths that excite a third fluorophore. A slider containing variousemission filters (not shown) is positioned beneath microscope (12A).Each emission filter specifically blocks the excitation wavelengthsemitted by a particular group of excitation lamps (such as (203) plus(204)) and allows the dimmer fluorescence to be seen via the microscope.Wired or wireless communication between the slider and the variousexcitation lamps is established so that the position of the slidercoordinates the correct excitation lamps to be energized based on theemission filter positioned between microscope (12B) and stage plate(200). By substituting a different stage plate (200), or changing therotational orientation of the stage plate, along with the slider oremission filters in the slider, it is possible to examine an arbitrarilylarge number of fluorophores.

FIG. 4 is a schematic of a power supply and lamp assembly in twodifferent configurations. In panel 4A, the typical configuration isshown, with a capacitor (400) inside the power supply to stabilize thepower supply's voltage output. In panel 4B, an improvement to protectthe LEDs is shown. When LEDs (401) are connected directly across a livepower supply, by direct connection of contacts (402), this is termed“hot-plugging. In panel 4B, the capacitor (400) is moved from the powersupply to the lamp assembly. This protects the LEDs (401) from currentin-rush during the time that the power supply adjusts its voltage toprovide a constant current level to the LEDs when hot-plugging.

FIG. 5 is a functional schematic comparing the prior artepi-illumination fluorescence dissecting stereomicroscope in panel 5Awith the present invention's independent direct-illuminationquick-change fluorescence system positioned below a standard dissectingstereomicroscope in panel 5B. In both 5A and 5B, an example is shownwhere a specimen (23) containing the fluorophore mCherry is illuminatedby pure yellow light and the yellow light is blocked from reaching theeyepiece, allowing much dimmer red fluorescent light emitted by thespecimen through to be observed. In both 5A and 5B, the microscopeoccupies the area of the dotted-line box and is represented by theEyepiece (12B) and the Objective lens (12C): (604) and (603),respectively. In both 5A and 5B, the quickly-changeable fluorescenceexcitation and detection element occupies the area of the dashed-linebox: (605) and (602), respectively. Note, in the prior art, that (605)interrupts the optical path of the microscope (604) and requires aspecial microscope that allows for this insertion; whereas, in thepresent invention, 5B, (602) is completely external to the microscope(603), so it can be used with any microscope model. In 5A, apolychromatic light source is external to and shines into module (605).The polychromatic light source is filtered by excitation filter (19),bounces off dichroic mirror (601), passes through objective lens (12C)and finally hits a specimen (23). In 5B, an integrated light source(101) that may have a spectrum more closely approximating the excitationspectrum of the specimen (23), is optionally refined by excitationfilter (19) and shines directly on the specimen (23) without any needfor a dichroic mirror or interruption of the optical path. In 5A,fluorescence emitted by specimen (23) passes through objective lens(12C), dichroic mirror (601) and emission filter (2) before reaching theeyepiece (12B). In 5B, fluorescence emitted by specimen (23) passesthrough emission filter (2) and then travels straight through the normaloptical path of the microscope from objective lens (12C) to eyepiece(12B).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENT

The current invention solves the problems associated with usingelectromagnetic radiation sources such as LEDs to excite and analyzefluorescence. Throughout this patent application, reference is made to“light”, Light Emitting Diodes (“LEDs”), and “fluorescence”; however, itis to be understood that “light” is a metaphor and meant to include allelectromagnetic wavelengths, including those which cannot be perceivedby the human eye, and “LEDs” are just one example of a source ofelectromagnetic radiation (others include but are not limited to gasdischarge lamps, x-ray and magnetron tubes, and a multitude of differenttypes of lasers), and “fluorescence”, though this word typically refersto the visible or invisible radiation emitted by certain substances as aresult of incident radiation of a shorter wavelength such as X-rays orultraviolet light, in this application is expanded and refers to anydetectable phenomenon that results from excited molecules, for exampleit could be a change in nuclear spin in response to radio waves in amagnetic field.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. All publications mentioned hereinare incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited. Further, the dates of publication provided may be different fromthe actual publication dates, which may need to be independentlyconfirmed. Before the present invention is described in further detail,it is to be understood that the invention is not limited to theparticular embodiments described, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

In addition to the use of single electromagnetic sources (for exampleLEDs) to excite atoms and/or molecules, it is possible to increasesignal, and the signal-to-noise ratio, through the use of multiplesingle-colored LEDs shone upon the sample from different angles (obliqueand/or orthogonal to the angle of observation), each with its ownoptical elements to focus and/or collimate it. Thus, the light from theLEDs does not need to be combined via complex optical elements, and noexpensive light pipe is needed, resulting in reduced cost, complexity,and increased optical efficiency and cost efficiency. Since multiplebeams converge at the point of interest in the observation focal plane,the excitation light is multiplied by the number of beams. Since thebeams each strike the point of interest from a different oblique angle(or in another preferred embodiment, orthogonal angle), the illuminationbeams do not converge behind or in front of the point of interest, sothe excitation light is not multiplied in other planes, resulting inreduced background fluorescence and an increase in the signal-to-noiseratio.

To solve the problem of quickly changing between fluorophores, themultitude of LEDs for each fluorophore and all associated optics, forexample lenses and filters, can be housed inside a single lamp assembly,such that any of a multiple of lamp assemblies can be slid into theactive position that illuminates the sample using a slider mechanism.Various multi-lamp assemblies can be quickly interchanged to createdifferent sets and different orders due to a unique magnetic attachmentmating structure that uses strong magnets and guide posts on themulti-lamp assemblies and matching magnets of the opposite polarity andguide holes on the slider. We present one of many possible embodimentswith a linear slide arrangement, but this does not exclude otherembodiments such as a rotational slide arrangement, or still others.Significantly, this slider system of multiple lamp assemblies eachcontaining elements to produce multiple-beams, is completely independentof the internal optical path of the microscope, so there is norequirement for a specially constructed microscope, and the currentinvention can be added on to any existing microscope. For simplicity andcost-effectiveness, the slider mechanism disclosed is hand-powered andhand-operated. Nevertheless, the slider must stop at precise locationsto align the optics, reproducibly. This is accomplished using strongmagnets in the slider and in a reference element (such as the slider'ssupporting holder), such that magnets of opposite polarity align at thepoints where the slider needs to stop, creating a detent system to indexthe slider's movement. Not only does this draw the slider to the correctposition and stop the slider, but also, in the event that the stoplocation is passed, it tends to draw the slider back into the correctstop location. Since only the stop locations of the preferred embodimentare magnetic or attracted by magnets, there is no force abrading orwearing the slider mechanism during the sliding operation (as comparedwith a typical spring-loaded ball/pin that is always under force,seeking a depression detent point).

Here are some features important in the construction of one preferredembodiment. Not every feature is required; in fact, using some featuresmay make other features obsolete or contraindicated:

One preferred embodiment is a system for the excitation and detection offluorescence, from various different fluorophores, underneath adissecting stereomicroscope. Different fluorophores require differentwavelengths of light to excite them, and then they emit differentwavelengths of light as fluorescence that must be detected/visualized.For example, a fluorophore originally derived from a jelly fish protein,called eGFP, is best excited by light at a peak wavelength of about 488nanometers (nm). eGFP then emits light at a peak wavelength of about 509nm. eGFP can be excited using an LED that has a dominant wavelength inthe neighborhood of about 470 nm because even “single-colored” LEDs tendto have a fairly broad spectrum. Even though an LED may be specified as“470 nm”, it emits some light all the way from 430 nm to 530 nm.Therefore, any excitation light with wavelengths found in the emissionspectrum, for example light that is over 500 nm, must be filtered out sothat it does not overwhelm the emission signal. This can be accomplishedwith a pair of filters. Light from the LED can be filtered so that onlylight below 490 nm (blue) is allowed to pass to the sample, and lightcoming from the sample can be filtered such that only light that is over500 nm (green) is allowed to pass on to the observer. Anotherfluorophore, named mCherry, is best excited by light with a wavelengthof about 587 nm and emits the most light at approximately 610nanometers. Appropriate LEDs, excitation filters, and emission filterscan be chosen to show mCherry fluorescence, as well. More specificinformation about LEDs, and their dominant wavelengths, spectra, andpower levels, etc. can be found in “Cree Xlamp XP-E2 LEDs” and thisinformation is hereby incorporated by reference. More information aboutvarious fluorophores useful in biology research can be found inCHUDAKOV, D. M. et al., “Fluorescent Proteins and Their Applications inImaging Living Cells and Tissues”, Physiological Reviews, AmericanPhysiological Society, 2010, Vol. 90, No. 3, Pages 1103-1163, and ishereby incorporated by reference.

FIG. 5 shows an important, novel aspect of the invention anddistinguishes it from prior art. A key feature of the invention is thatit takes place independently and/or externally to any other piece ofequipment. For example, if a microscope is used to observe fluorescence,the invention is located outside of the microscope and is not dependenton interrupting the optical path of the microscope to excite thespecimen or to filter the emission from the specimen. FIG. 5 is afunctional schematic comparing the prior art epi-illuminationfluorescence dissecting stereomicroscope in panel 5A with the presentinvention's independent direct-illumination quick-change fluorescencesystem positioned below a standard dissecting stereomicroscope in panel5B. In both 5A and 5B, an example is shown where a specimen (23)containing the fluorophore mCherry is illuminated by pure yellow lightand the yellow light is blocked from reaching the eyepiece, allowingmuch dimmer red fluorescent light emitted by the specimen through to beobserved. In both 5A and 5B, the microscope occupies the area of thedotted-line box and is represented by the Eyepiece (12B) and theObjective lens (12C): (604) and (603), respectively. In both 5A and 5B,the quickly-changeable fluorescence excitation and detection elementoccupies the area of the dashed-line box: (605) and (602), respectively.Note, in the prior art, that (605) interrupts the optical path of themicroscope (604) and requires a special microscope that allows for thisinsertion; whereas, in the present invention, 5B, (602) is completelyexternal to the microscope (603), so it can be used with any microscopemodel. In 5A, a polychromatic light source is external to and shinesinto module (605). The polychromatic light source is filtered byexcitation filter (19), bounces off dichroic mirror (601), passesthrough objective lens (12C) and finally hits a specimen (23). In 5B, anintegrated light source (101) that may have a spectrum more closelyapproximating the excitation spectrum of the specimen (23), isoptionally refined by excitation filter (19) and shines directly on thespecimen (23) without any need for a dichroic mirror or interruption ofthe optical path. In 5A, fluorescence emitted by specimen (23) passesthrough objective lens (12C), dichroic mirror (601) and emission filter(2) before reaching the eyepiece (12B). In 5B, fluorescence emitted byspecimen (23) passes through emission filter (2) and then travelsstraight through the normal optical path of the microscope fromobjective lens (12C) to eyepiece (12B). Compared to the prior art, thepreferred embodiment completely dispenses with the dichroic mirror (601)and the excitation light passes through two fewer optical elements.

In order to get enough light to effectively excite mCherry, multipleLEDs with a dominant output wavelength of about 585 nm, need to becollimated into beams, or focused to points. Light, which diverges froman LED source, can be captured and collimated using a typical opticalmeans for that purpose, for example, a non-exhaustive list includes aplano-convex lens, a biconvex lens, a Fresnel lens, a reflector, anoptic that makes use of total internal reflectance, or any combinationthereof, to name a few possibilities, placed in front of the LED. Thiscollimated beam can then be restricted efficiently, by a dichroic filterto below 600 nm. More specific and useful information about dichroicfilter technology is found in Macleod, H. A. “Thin Film Optical Filters”and the entirety of this teaching is hereby incorporated by reference.

In biology research, it is often desirable to track the expression ofone gene in relation to the expression of at least one other gene as areference. Therefore, it is desirable to be able to analyze theexpression of two or more fluorophores, in the same live, movingspecimen, in rapid sequence. In some cases, the fluorophore expressionwill co-localize and it is important to be able to excite and detecteach fluorophore individually. Therefore, a preferred embodimentincludes one excitation module that emits electromagnetic radiation (inthis case blue light) that is restricted to be less than 495 nanometersto excite a fluorophore such as eGFP without exciting a fluorophore suchas mCherry, and a second excitation module that emits electromagneticradiation (in this case green and/or yellow light) that is restricted tobe greater than about 510 nanometers to excite a fluorophore such asmCherry without exciting a fluorophore such as eGFP.

Each filtered beam can be aimed at the same point on the sample planesuch that the beams intersect at that point. This results in a point, inthe sample plane, where the brightness is multiplied by the number ofLEDs. However, the point proximal to that point, and the point distal tothat point do not have the same brightness multiplication, because thebeams do not converge in other planes. This results in a significantdecrease in background fluorescence, and an increase in thesignal-to-noise ratio for the fluorophore present in the sample ofinterest. An example device to practice this method is shown in FIG. 1 ,where (1) is an enclosure pertaining to Light Emitting Diodes (LEDs)(101), (102), and (103). The light rays from these LEDs are collimatedby optics (104), (105), and (106), respectively, and filtered by filters(19), (108), and (20), respectively. The resulting light beams, (110),(111), and (112), respectively, intersect and add at the observationplane (114), but not in planes in front of (for example, (113)) orbehind (for example (115)) the sample plane of interest.

The bright spot of excitation light is removed from the visual field ofthe observer using another filter, such as a dichroic filter. The secondfilter does not allow the excitation wavelength through, but does allowthe emission wavelength through. In the preferred embodiment, all of theoptical elements described above are combined into a single relativelycompact enclosure plus attached emission filter support. Means is alsoprovided for the LEDs in such an enclosure to be energized selectivelybased on an external signal. This can be accomplished via slidingelectrical contacts, inductive power coupling, switches, sensors, andother methods.

The preferred embodiment may include a slider mechanism that can beeasily attached to structures just past the objective lens(es) of anymicroscope system. A multitude of the lamp assemblies can be attached toa linear slider of arbitrary length, or a rotational slider of arbitrarydiameter. The preferred embodiment also employs a magnetic system foraligning the slider, and thus the lamp systems with their filters,between the objective lens and the sample, with high precision. Modulesfor eGFP, and for mCherry, and for a multitude of other fluorophores canbe easily attached to the slider mechanism in the preferred embodiment.The user can switch between analyses of these different fluorophoressimply by sliding the module of interest into the active position, usingthe slider. FIG. 2 and FIG. 3 provide examples.

FIG. 2 represents one preferred embodiment of a High-efficiency ObliqueIllumination System for Fluorescence Excitation and Detection for amulti-fluorophore fluorescence microscope system. Multi-LED lampenclosure (1), with excitation filters (19) and (20) is furtherassembled to emission filter (2); similarly, multi-LED lamp enclosure(3), with excitation filters (21) and (22), different from (19) and(20), is further assembled to a different emission filter (4). Thesecombined assembly modules of excitation light sources with emissionfilters are detachably attached to sliding chassis (5). Astereo-microscope is represented by main body (12A), eyepieces (12B) andobjective lens (12C). Slider-holder (10) positions the slider (5) in theoptical path of the microscope. Through the use of handles (17) and(19), slider (5) can be translated in directions (18) and (20) by eitherpushing action or pulling action, such that either lamp/filterassemblies (1) plus (2) or (3) plus (4) are positioned below the opticalpath of the microscope and electrical contacts are established toenergize the lamps within them via contacts (13) plus (14) or (15) plus(16), respectively. Optimal positioning of the slider is insured by theattractive force of slider detent magnets (6), (7), and (8) withstationary reference magnet (9) located in the slider-holder (10). Themulti-LED lamps have beams aimed to converge at point (23) in the focalplane of the microscope. The spot (23) is also covered by the matchingemission filter. Pivot screws (24) and (25) allow lamp enclosures (1)and (3), respectively, to pivot to precisely illuminate spot (23) whenin the active position. For example, lamp enclosure (1) is in the activeposition when slider magnet (6) aligns with stationary reference magnet(9). Not shown in this figure, because they are blocked from view byslider (5), are adjustable jack screws coming toward the slider fromenclosures (1) and (3) The heads of these jack screws hit slider (5) sothat the tilt angles of lamp enclosures (1) and (3) to can be adjustedto precisely illuminate spot (23) when in the active position.

Though LEDs represent one of the most energy-efficient forms of lightgeneration, high-brightness LEDs still generate considerable heat thatmust be dissipated to prevent malfunctions. One preferred embodimentaccomplishes this thermal dissipation by thermally coupling the LEDs tothermally conductive aiming mechanisms, which are in turn coupled to athermally conductive enclosure.

The preferred embodiment requires means to accurately aim theconvergence spot of the multi-LED lamp assemblies at the same point inthe center of the microscope's field of view. This can be accomplishedby varying the angle of the lamp in two polar axes, often termed thetaand phi. Left-right aiming can be accomplished by rotating the lampassembly (yaw in the theta axis) when it is attached to its bracket by asingle screw located at the left-right center, provided that means areprovided for sufficient friction to prevent the lamp from rotating dueto accidental bumping, vibration, inertia, etc. Elements (24) and (25)of FIG. 2 provide an example. Up-down aiming (pitch adjustment in thephi axis) can be accomplished by use of a jack screw that pushes againsta hinge that is under constant force due to, for examples, gravity or aspring. Within each multi-LED lamp assembly, each LED must be aimed sothat its beam converges at the same spot. This aiming requires highprecision because the LEDs and optical elements are small. The preferredembodiment provides sliding and rotating degrees of freedom for eachLED's aiming support that glide on thermally conductive grease.

While it is possible for two hard items to slide against each other,this generally causes abrasion and wear. The preferred embodimentprevents such wear by making the slider of a bearing material, such asPolytetrafluoroethylene, Polyoxymethylene, etc. and the slider's holderout of a hard, durable material such as anodized aluminum or stainlesssteel. An alternate preferred embodiment swaps the above material typesbetween the slider and its slider holder. As mentioned earlier, apreferred embodiment uses magnets to stop the slider or turret at pointswhere modules are aligned to excite and analyze a specimen; therefore,neither the slider-holder nor the slider can be made from aferromagnetic material.

Because there are a multitude of fluorophores, each having its ownspecific excitation and emission wavelengths, a multitude of lamp/filterassemblies are needed, and it is desirable for users to be able toexchange the assemblies quickly and conveniently. In one preferredembodiment, this is accomplished through magnetic attachment so that notools or conventional fasteners are needed. Each module includes abracket with two strong magnets and two guide posts. The slider thatreceives the modules has two guide holes in locations spaced to receivethe two module guide pins, and two magnets of opposite polarity toattract and hold the module's magnets creating a mating structure. Thusno tools are required to install, remove, or change the modules.Alternatively, one or multiple guide post(s) can be exchanged to guidehole(s) and magnetic polarities can be changed in any arrangementallowing for attachment of the modules to the slider. So that users canquickly identify which module is associated with a particularfluorophore, the modules are color-coded with the color of thefluorescence emission. For example, the module that is used for excitingand detecting Green Fluorescent Protein is colored green and the modulethat is used for exciting and detecting mCherry protein is coloredcherry-red.

To fit the available geometry of dissecting-type stereomicroscopes, itis desirable that modules can deliver small collimated spots of light ata distance in the range of 20 millimeters to 200 millimeters. Thepreferred embodiment accomplishes this with a pair of convex lenses witha combined focal length in the range of 20 millimeters to 200millimeters. Alternative preferred embodiments use optical elementsbased on the principle of Total Internal Reflectance or reflection aloneor in combination with refraction.

A preferred embodiment for quickly changing between fluorophores, usingorthogonal, rather then oblique, excitation illumination is shown inFIG. 3 . In this case, it is geometrically simpler to physicallyseparate the excitation sources from the emission filters; however theystill communicate via wires or wirelessly so that appropriate excitationsources are energized, based on the emission filter chosen by the user.FIG. 3 is a drawing of an alternate embodiment of the invention withmultiple lamps of various colors aimed at the sample of interest,orthogonally with respect to the axis of observation. A microscope,represented by its body (12A) and eyepiece tubes (12B) is focused at alocation (209) where a specimen of interest would be in the center ofthe field of view of a stage plate (200). Excitation sources (203) and(204) project electromagnetic energy along intersecting beams (210) and(211) that intersect at the sample location (209). These beams are bothat wavelengths that excite a particular fluorophore. Similarly,excitation sources (205) and (206) are both at wavelengths that excite asecond fluorophore, and excitation sources (207) and (208) are both atwavelengths that excite a third fluorophore. A slider containing variousemission filters (not shown) is positioned beneath microscope (12A).Each emission filter specifically blocks the excitation wavelengthsemitted by a particular group of excitation lamps (such as (203) plus(204)) and allows the dimmer fluorescence to be seen via the microscope.Wired or wireless communication between the slider and the variousexcitation lamps is established so that the position of the slidercoordinates the correct excitation lamps to be energized based on theemission filter positioned between microscope (12B) and stage plate(200). By substituting a different stage plate (200), or changing therotational orientation of the stage plate, along with the slider oremission filters in the slider, it is possible to examine an arbitrarilylarge number of fluorophores.

One preferred embodiment directly energizes LEDs in the lamp assembliesby sliding electrical contacts on the lamp assembly against poweredelectrical contacts that are stationary on the slider holder. Thispresents a problem for conventional constant-current power supplydesigns because there is a high instantaneous in-rush of electricalcurrent before the power supply can sense the voltage needed for propercurrent. This current in-rush can damage LEDs. In a preferredembodiment, this problem is dealt with inexpensively by relocatingcapacitors that stabilize the output of the power supply from the powersupply to the lamp assemblies as shown in FIG. 4 . FIG. 4 is a schematicof a power supply and lamp assembly in two different configurations. Inpanel 4A, the typical configuration is shown, with a capacitor (400)inside the power supply to stabilize the power supply's voltage output.In panel 4B, an improvement to protect the LEDs is shown. When LEDs(401) are connected directly across a live power supply, by directconnection of contacts (402), this is termed “hot-plugging. In panel 4B,the capacitor (400) is moved from the power supply to the lamp assembly.This protects the LEDs (401) from current in-rush during the time thatthe power supply adjusts its voltage to provide a constant current levelto the LEDs when hot-plugging. More information about this is includedin the cited reference by Cree, Inc., “LED Electrical Overstress”, theentirety of which is hereby incorporated by reference.

The above listed features taken all together might seem to be acomplicated invention, but when taken in meaningful subsets, the noveltyof the invention becomes clear. Specific methods and devices of aHigh-efficiency Oblique Illumination System for Fluorescence Excitationand Detection have been disclosed. It should be apparent, however, tothose skilled in the art that many more modifications besides thosealready described are possible without departing from the inventiveconcepts herein. The inventive subject matter, therefore, is not to berestricted to the examples mentioned in the disclosure. Moreover, ininterpreting the disclosure, all terms should be interpreted in thebroadest possible manner consistent with the context. In particular, thewords should be interpreted as referring to elements, components, orsteps in a non-exclusive manner, indicating that the referencedelements, components, or steps may be present, or utilized, or combinedwith other elements, components, or steps that are not expresslyreferenced.

I claim:
 1. A module for electromagnetic projection and detection offluorescence comprising: at least two sources of electromagneticradiation; said sources of electromagnetic radiation shone directly atessentially the same point; said sources of electromagnetic radiationshone at said point, via at least two distinct 3-dimensional angles, inlines not parallel to the line between a point of observation and aspecimen; a detection element disposed across said line between a pointof observation and a specimen, said element operating on a spectrumassociated with the spectrum of the electromagnetic radiation sources;at least one of: sliding electrical contacts, inductive power couplings,sensors, wired communication, and wireless communication; toconcomitantly energize said electromagnetic radiation source; and devoidof a dichroic mirror.
 2. A module as in claim 1 wherein said element isa filter disposed to block electromagnetic radiation from said sourcesand to transmit electromagnetic energy emitted by said specimen.
 3. Amodule as in claim 1 wherein at least one of: said sources ofelectromagnetic radiation are of essentially the same spectrum; and saidsources of electromagnetic radiation are filtered to refine theirspectra.
 4. A module as in claim 1 wherein at least one of: said sourcesof electromagnetic radiation have individual optics with the ability tofocus near said specimen; and said sources of electromagnetic radiationhave individual collimation optics with the ability to direct a beamtoward said specimen.
 5. A module as in claim 1 wherein: said anglescause less electromagnetic radiation intensity per unit area to bepresent in orthogonal planes other than the orthogonal plane containingsaid specimen.
 6. A module as in claim 1 wherein: said element blocksthe spectrum of electromagnetic radiation emitted by said sources andpasses a different spectrum emitted by said specimen in response to theelectromagnetic radiation emitted by said sources.
 7. A module as inclaim 1 wherein: said module is disposed for further assembly to amoveable chassis creating a moveable multiple wavelength excitation anddetection assembly.
 8. A module as in claim 1 wherein: said sources emitlight of less than about 495 nanometers wavelength; and said elementpasses light of greater than about 500 nanometers wavelength.
 9. Amodule for the excitation and detection of fluorescence comprising: atleast one electromagnetic radiation filter disposed to be moved into apath between a sample and a point of observation, for detection; atleast one electromagnetic radiation source disposed to excite saidsample from a point outside said path between said sample and said pointof observation; means to concomitantly energize said electromagneticradiation source responsive to positioning said electromagneticradiation filter inside said path between a sample and a point ofobservation wherein said electromagnetic radiation filter and saidelectromagnetic radiation source are physically linked, automatically;and devoid of manual switching of said electromagnetic radiation sourcerelative to said electromagnetic radiation filter.
 10. A module as inclaim 9 devoid of a dichroic mirror.
 11. A module as in claim 9 furthercomprising a mating structure for the precise removable attachment ofsaid module at at least one possible position on a moveable chassis viainteraction with a reciprocal mating structure located on said chassis.12. A module as in claim 11 wherein said mating structure furthercomprises at least one of a magnet; and a ferromagnetic material.
 13. Amodule for the excitation and detection of fluorescence comprising: atleast one electromagnetic radiation filter disposed to be moved into apath between a sample and a point of observation, for detection; atleast one electromagnetic radiation source disposed to excite saidsample from a point outside said path between said sample and said pointof observation; at least one of: sliding electrical contacts, inductivepower couplings, sensors, wired communication, and wirelesscommunication to concomitantly energize said electromagnetic radiationsource responsive to positioning said electromagnetic radiation filterinside said path between said sample and said point of observation; andsaid electromagnetic radiation filter in fixed physical relationshipwith said radiation source for automatic coordination of said sourcewith said filter.